Leveraging engineered Pseudomonas putida minicells for bioconversion of organic acids into short-chain methyl ketones

Methyl ketones, key building-blocks widely used in diverse industrial applications, largely depend on oil-derived chemical methods for their production. Here, we investigated bio-based production alternatives for short-chain ketones, adapting the solvent-tolerant soil bacterium Pseudomonas putida as a host for ketone biosynthesis either by whole-cell biocatalysis or using engineered minicells, chromosome-free bacterial vesicles. Organic acids (acetate, propanoate and butyrate) were selected as the main carbon substrate to drive the biosynthesis of acetone, 2-butanone and 2-pentanone. Pathway optimization identified efficient enzyme variants from Clostridium acetobutylicum and Escherichia coli, which were tested under both constitutive and inducible expression of the cognate genes. By implementing these optimized pathways in P. putida minicells, which can be prepared through a simple 3-step purification protocol, the feedstock was converted into the target short-chain methyl ketones, remaining catalytically functional for >4 months. These results highlight the value of combining morphology and pathway engineering of non-canonical bacterial hosts to establish alternative bioprocesses for toxic chemicals that are difficult to produce by conventional approaches. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/574483v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@14db69eorg.highwire.dtl.DTLVardef@1104c0corg.highwire.dtl.DTLVardef@f0c725org.highwire.dtl.DTLVardef@1a28c2b_HPS_FORMAT_FIGEXP M_FIG C_FIG